The pharmaceutical sector is currently experiencing efforts in rethinking the way research and development can be performed more efficiently. One important issue that needs to be addressed is the lack of efficient and reproducible drug discovery models that are able to predict toxicity and efficiency of compounds in humans prior to launch of expensive clinical trials.
Similarly, chemical companies as well as public and regulation authorities are looking for alternative in-vitro methods to animal testing. The latter poorly predict the human response, are ethically controversial and costly. However, the absence of efficient and reproducible in-vitro models able to predict the toxicity of chemical compounds in humans is one hurdle that needs to be tackled in toxicology testing.
This is particularly true for in-vitro models of the lung, due to the complex cellular microenvironment present in the lower airways where the gas exchange takes place. The in-vivo conditions of the lungs, such as air-liquid interface, respiratory movements, shear stresses induced by liquids over the epithelial layer and at the endothelium, etc. are particularly complex, which is a reason why accurate in-vitro alveolar models do not exist to date.
Known in-vitro models of the lung, such as transwell systems, can reproduce tissue interfaces between alveoli and vascular endothelium, but neither the mechanical processes of physiological breathing nor the shear stress induced by the blood stream, nor the removal of the soluble products and the waste products excreted by the cells. In addition, transwell membrane thicknesses are typically between ten and twenty micrometers, which is about one to three orders of magnitude larger than the in-vivo dimension. This can importantly hamper the epithelial endothelial signalling.
In this context, research platforms based on microfluidic technologies are currently emerging and have the potential to improve in-vitro model accuracy and experimental efficiency. For example, microfabricated bio-artificial lung models have only been reported in the past three years.
In WO 2010/009307 A2 a breathing lung-on-chip is described using a co-culture of epithelial/endothelial cells. This lung-on-chip is made of two superposed microchannels separated by a thin porous and stretchable membrane made of Polydimethylsiloxane (PDMS). This membrane, on which epithelial and endothelial cells are cultured, is cyclically linearly stretched by varying the pressure in adjacent channels, which allows the deformation of the walls of the microchannels on which the thin porous membrane is attached.
However, the actuation principle shown in WO2010/009307 A2 suffers important limitations. Indeed, the accuracy of the stress level of the thin membrane directly depends on the amplitude of the deflection of the internal channel walls, which in turn is a function of a number of parameters, in particular, the mechanical properties and the geometry of the walls material and the actuation pressure, which all need to be accurately controlled. In addition, the construction of the device based on the assembly of two parts—the top and the bottom parts—between which the thin membrane is sandwiched requires an accurate alignment to guarantee the batch to batch reproducibility of the mechanical properties of the channel walls.
At least these factors do not allow the precise control of the stretching level of the thin membrane and ultimately of the cells that are cultured on this membrane. Therefore, the fabrication processes of the device must be extremely accurate, which increases the production costs and/or may require a costly calibration of the stress in the membrane in function of the applied pressure for each device. Furthermore, several aspects of this device only approximately reproduce the basement membrane of the lung alveoli and its deformation. Indeed, the unidirectional stretching generated by the adjacent channels of this device does not correspond to the three-directional stretching that takes place in the human lung. In-vivo, the respiratory movements are the result of the contraction of the diaphragm that pulls the cavity of the lung, causing air to enter in the lungs. Also, the membrane integrated in the lung-on-chip described in WO2010/009307 A2 is comparably thick, i.e., about ten micrometers, as compared to the thickness of the basement of the lung alveolar membrane, which is between 200 and 500 nanometers.
In US 2010/0233799 A1 another example of a microfluidic lung-on-chip with a stretchable membrane is shown. Thereby, a device includes a PDMS membrane on which epithelial cells are cultured. In order to investigate the mechanical stresses that typically occur in ventilated lungs, a pin exerts a mechanical force on the membrane.
However, for being suitable to such pin exertion, the membrane has to be robust and is in the device of US 2010/0233799 A1 about 100 micrometers thick. The shown device is not equipped with a porous membrane and thus does not allow mimicking the complexity of the alveolar membrane by reproducing the air-blood barrier. Further, the device does not allow for the culture of cells at the side of the membrane where the pin pushes to deform the membrane since the direct contact of the pin with the membrane would squeeze the cells and damage them. This means that this system does not allow mimicking the co-culture system typical to in-vitro barriers, even if one would integrate a porous membrane.
An additional limitation of the system described in US 2010/0233799 A1, also due to the direct contact of the pin against the membrane, is the obvious absence of space between the pin and the membrane. This prevents providing sufficient space for physiological medium, and as a consequence, cells could not be perfused. Since no physiological solution can be provided to the cells from the bottom of the membrane, the physiological medium needs to be provided from the top of the membrane where the cells are cultured, meaning that no air-liquid interface (ALI) condition can be set up in this system.
Another downside of the system of US 2010/0233799 A1 is that the observation of the cells from the bottom of the chip is not possible with a non-transparent pin. And even if the pin were transparent, the images would still be distorted due to the curvature of the pin, and thus would have to be corrected, e.g., by a specific software, in order to be appropriate.
Still further, the system of US 2010/0233799 A1 only allows stretching the membrane in one outward direction, whereas in the lung, this direction is only true for the endothelial cells. Also, the stretch profile in the system of US 2010/0233799 A1 is very heterogeneous. In particular, only the membrane in the middle will adapt to the structure of the pin, whereas the membrane in the periphery does not adapt. This results in different stretch profiles between the middle of the membrane and the periphery of the membrane and, in addition, the alveoli in the lung are stretched similar to an expanding sphere. This means that the radius of the sphere changes constantly wherein, in this system, the radius is given by the shape of the pin.
Therefore, there is a need for a device for in-vitro modelling tissues of organs that allows to, e.g., cyclically stretch cells in predefined and varying extent and/or direction thus making it possible to mimic three-dimensional deformation of the tissue such as the lung alveoli.